Jungmook Kim

Main conclusion Activating and repressive promoter elements and the 5′ UTR coordinately regulate spatiotemporal expression of the male gametophyte-specific transcription factor LBD27 during pollen development in Arabidopsis. Abstract The Lateral Organ Boundaries Domain (LBD) family consists of plant-specific transcription factors that regulate diverse aspects of growth and development. In Arabidopsis thaliana, SIDECAR POLLEN/LATERAL ORGAN BOUNDARIES DOMAIN 27 (SCP/LBD27) is a male-gametophyte-specific LBD transcription factor that controls asymmetric division and cell fate specification during early pollen development. Although precise spatiotemporal expression of LBD27 is essential for its developmental function, the molecular mechanisms and cis-regulatory elements underlying this regulation remain largely unknown. In this study, we investigated the regulatory elements that govern LBD27 expression during pollen development by dissecting its promoter and 5′ untranslated region (UTR). Analysis of a series of LBD27 promoter deletion constructs fused to GUS:eGFP or histone H2B:eGFP reporters showed that the 5′ UTR is necessary for expression in both the vegetative cell nucleus and sperm cell nuclei during pollen development. In addition, a distal promoter region spanning −1501–−801 bp contains regulatory elements that repress LBD27 expression at later stages of pollen development. Together, these results suggest that the spatiotemporal expression of LBD27 is regulated through combinatorial interactions between distinct promoter regions and the 5′ UTR, thereby ensuring precise control during male gametophyte development in Arabidopsis.

Small signaling peptides play pivotal roles in coordinating plant growth, development, and immunity. In Arabidopsis, ROOT MERISTEM GROWTH FACTOR 1 (RGF1) and its cognate receptor RGF1 INSENSITIVE 1 (RGI1) suppress lateral root (LR) development by activating the transcription factor gene PUCHI via mitogen-activated protein kinase signaling. WRKY30 has previously been identified as an early gene strongly induced by RGF1; however, its role in LR regulation remains unclear. Here, we demonstrate that WRKY30 functions downstream of the RGF1–RGI1 module to inhibit LR development. In ProWRKY30:GUS and ProWRKY30:SVnlsTomato lines, RGF1 treatment strongly induced reporter expression in the distal region of primary roots, while this response was markedly reduced in the rgi1 mutant. Furthermore, RGF1 enhanced WRKY30 promoter-fused β-glucuronidase (GUS) activity during LR development. Overexpression of WRKY30 under the UBIQUITIN10 or RGI1 promoters suppressed LR initiation in wild-type and rgi1 plants without affecting RGF1-promoted root meristem activity. Conversely, CRISPR/Cas9-generated wrky30 mutants exhibited reduced sensitivity to RGF1-mediated suppression of LR formation. Collectively, these findings indicate WRKY30 as a key downstream component of RGF1–RGI signaling that specifically restricts LR development.

The intricate regulation of gene expression determining cell fate during male gametogenesis involves a complex interplay of multiple transcriptional regulators. In Arabidopsis (Arabidopsis thaliana), the LATERAL ORGAN BOUNDARIES DOMAIN 10 (LBD10) transcription factor is prominent in early microspores and both the germ and vegetative cells of bicellular pollen, playing an important role in pollen development. However, in mature pollen, LBD10 exclusively localizes in the vegetative cell nucleus. Here, we identify cis-acting elements and trans-acting factors responsible for the specific expression of LBD10 in the vegetative cell nucleus during pollen maturation. Using a series of LBD10 promoter deletion constructs fused with GUS or GFP reporters, we pinpoint two crucial core promoter sequences. These sequences are situated within two 200 bp regions upstream of the start codon and independently govern LBD10 expression in the vegetative cell nucleus. We demonstrate that a W-box motif (AGTCAC) at –770 bp is essential for activating the expression of LBD10 in vegetative cells during pollen maturation. Our transient gene expression assays using Arabidopsis protoplasts and chromatin immunoprecipitation assays show that the transcription factors WRKY2 and WRKY34 recognize the LBD10 promoter region containing W-box motifs. Collectively, our findings suggest that WRKY2 and WRKY34 binding to the W-box motifs plays a role in the vegetative cell nucleus-specific expression of LBD10 in pollen. This interaction may contribute to male gametophyte development, shedding light on the intricate regulatory network governing this critical biological process.

Summary: Reproduction in angiosperms relies on the precise growth of pollen tubes, facilitating the delivery of sperm cells to the ovule for double fertilization. LATERAL ORGAN BOUNDARIES DOMAIN10 (LBD10), a plant‐specific transcription factor, plays a pivotal role in Arabidopsis pollen development. Here, we uncovered LBD10's function in sustaining pollen tube growth and integrity. The lbd10 mutant exhibited elevated levels of reactive oxygen species (ROS) and hydrogen peroxide (H₂O₂) in both pollen grains and tubes, leading to compromised pollen tube growth. The inhibition of ROS synthesis and scavenging of excess ROS with an antioxidant treatment each alleviated these defects in lbd10. The lbd10 mutant displayed reduced flavonol accumulation in both pollen grains and tubes. All the altered phenotypes of lbd10 were complemented by expressing LBD10 under its native promoter. Exogenous application of flavonoids recused the defects in pollen tube growth and integrity in lbd10, along with reducing the excess levels of ROS and H₂O₂. LBD10 directly binds the promoters of key flavonol biosynthesis genes in chromatin and promotes reporter gene expression in Arabidopsis mesophyll protoplasts. Our findings indicate that LBD10 modulates ROS homeostasis by transcriptionally activating genes crucial for flavonol biosynthesis, thereby maintaining pollen tube growth and integrity.

Cytokinins regulate plant growth, development, and responses to environmental stresses such as cold via phosphorelay from cytokinin receptors to the ARABIDOPSIS RESPONSE REGULATORs (ARRs). However, the molecular mechanisms underlying the activation of type-B ARR transcriptional activity in Arabidopsis (Arabidopsis thaliana) remain unclear. Here, we show that the E3 SUMO ligase HIGH PLOIDY2 SUMOylates ARR1, a type-B ARR, at K236, triggering its activation. Cold- or cytokinin-induced phosphorylation of ARR1 at D89 is crucial for its interaction with HPY2. Lysine 236 is critical for ARR1's transactivation without compromising its DNA-binding ability, while D89 is crucial for ARR1's binding to target gene promoters. Cytokinin enhances ARR1's chromatin binding, but cold does not. ARR1 K236 plays a critical role in promoting histone H3 acetylation in response to both cytokinin and cold without affecting chromatin binding. The K236R mutation in ARR1 reduces target gene expression and alters cytokinin and cold response phenotypes. This study unveils a mechanism of ARR1 activation wherein phosphorylated ARR1 interacts with HPY2 and binds to chromatin in response to cytokinin. Cold triggers a phosphorelay targeting chromatin-bound ARR1. HPY2 then catalyzes ARR1 SUMOylation at K236, enhancing histone H3 acetylation and leading to transcriptional activation of ARR1 in response to both cold and cytokinin.

Summary: Chromatin configuration is critical for establishing tissue identity and changes substantially during tissue identity transitions. The crucial scientific and agricultural technology of in vitro tissue culture exploits callus formation from diverse tissue explants and tissue regeneration via de novo organogenesis. We investigated the dynamic changes in H3ac and H3K4me3 histone modifications during leaf‐to‐callus transition in Arabidopsis thaliana. We analyzed changes in the global distribution of H3ac and H3K4me3 during the leaf‐to‐callus transition, focusing on transcriptionally active regions in calli relative to leaf explants, defined by increased accumulation of both H3ac and H3K4me3. Peptide signaling was particularly activated during callus formation; the peptide hormones RGF3, RGF8, PIP1 and PIPL3 were upregulated, promoting callus proliferation and conferring competence for de novo shoot organogenesis. The corresponding peptide receptors were also implicated in peptide‐regulated callus proliferation and regeneration capacity. The effect of peptide hormones in plant regeneration is likely at least partly conserved in crop plants. Our results indicate that chromatin‐dependent regulation of peptide hormone production not only stimulates callus proliferation but also establishes pluripotency, improving the overall efficiency of two‐step regeneration in plant systems.

Leucine‐rich repeat receptor kinases (LRR‐RKs) play a pivotal role in diverse aspects of growth, development, and immunity in plants by sensing extracellular signals. Typically, LRR‐RKs are activated through the ligand‐induced interaction with a SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) coreceptor, triggering downstream signaling. ROOT MERISTEM GROWTH FACTOR1 (RGF1) INSENSITIVEs (RGIs) LRR‐RLK receptors promote primary root meristem activity while inhibiting lateral root (LR) development in response to RGF peptide. In this study, we employed rapamycin‐induced dimerization (RiD) and BAK1‐INTERACTING RECEPTOR‐LIKE KINASE3 (BIR3) chimera approaches to explore the gain‐of‐function of RGI1, RGI4, and RGI5. Rapamycin induced the association of cytosolic kinase domains (CKDs) of RGI1 and the BAK1 coreceptor, activating both mitogen‐activated protein kinase 3 (MPK3) and MPK6. Rapamycin significantly inhibited LR formation in RiD‐RGI1/RGI4/RGI5‐BAK1 plants. Using transgenic Arabidopsis expressing RGI1CKD fused to the BIR3‐LRR chimera under estradiol control, we observed a substantial reduction in LR density upon β‐estradiol treatment. Additionally, we identified a decrease in root gravitropism in BIR3 chimera plants. In contrast, RiD‐RGI/BAK1 plants did not exhibit defects in root gravitropism, implying the importance of combinatorial interactions between RGIs and SERK coreceptors in the inhibition of root gravitropism. Constitutive activation of RGIs with BAK1 in RiD‐RGI/BAK1 plants by rapamycin treatment resulted in the inhibition of primary root growth, resembling the inhibitory effects observed with high concentrations of phytohormones on primary root elongation. Our findings highlight that the interactions between CKDs of RGIs and BAK1, constitutively induced by rapamycin or BIR3 chimera, efficiently control LR development.

Leucine-rich repeat-receptor kinases (LRR-RKs) perceive various endogenous peptide hormones that control plant growth and development. However, the majority of corresponding ligands and their direct ligand-binding receptors have not been identified yet. A recent study demonstrated that three LRR-RK PLANT PEPTIDE CONTAINING SULFATED TYROSINE RECEPTORS (PSYRs) act as ligand-receptors of the PSY family peptides that mediate the trade-off between the optimal plant growth and stress tolerance responses. The genetic, biochemical, and transcriptome analyses suggested that PSYR1, PSYR2, and PSYR3 function as negative regulators of plant growth in the absence of PSY peptides and induce stress tolerance responses, whereas the PSY family peptides repress PSYR signaling, allowing plant growth. This trade-off mechanism between plant growth and stress responses mediated by the PSY-PSYR signaling module allows plants to survive under ever changing environmental stresses.